According to the World Health Organisation, hepatitis C virus (HCV) infects approximately 170 million to 200 million people worldwide. While governments have increased education about how HCV is transmitted, and despite prevention programs, HCV continues to proliferate. Approximately 80% of those who are infected with HCV remain carriers of the virus. In Australia about 16,000 new cases of HCV infection are reported each year, the new infections being most prevalent amongst injection drug users. HCV is the most common blood-borne viral infection, causing the death of a substantial proportion of the population.
HCV is known to infect the liver and certain immune cells of a sufferer. As a result, HCV leads to serious liver disease such as fibrosis, cirrhosis, steatosis and heptocellular carcinoma (liver cancer) more frequently than other forms of hepatitis. HCV is a leading cause for the requirement of liver transplants. It is generally believed that the acute phase of the infection is often unrecognised due to the sub-clinical nature of the infection, and 80% of individuals progress to a chronic condition. Chronic infection is a result of the immune system's failure to generate an adequate immune response against the virus.
Currently there is no vaccine for HCV and the only available therapy for treatment of HCV has relied on development of antiviral drugs and drug combinations. The general idea behind antiviral drug design is to identify viral proteins, or parts of proteins, that can be disabled or inhibited. A standard treatment of choice for patients suffering moderate or severe fibrosis includes a combination of alpha-interferon and ribavirin. The antiviral effects of combination alpha-interferon and ribavirin therapy cause a rapid decrease in HCV levels in the blood, even after a single dose. Conventional alpha-interferon treatment for HCV however suffers several drawbacks. For example, (i) when alpha-interferon treatment is stopped after a few weeks or months of treatment, the viral load level is known to re-establish rapidly; (ii) treatment with alpha-interferon/ribavirin is associated with severe side effects, including flu-like symptoms, reduced red or white cell counts, bone marrow suppression, neuropsychiatric effects, particularly depression and anemia; (iii) effective treatment requires patient adherence to a frequent dosing regimen since alpha-interferon is absorbed and eliminated from the body rapidly; and (v) high cost of such treatments.
Some of the above drawbacks, referring particularly to item (iii) above, have been addressed by subjecting alpha-interferon to ‘pegylation’ in which polyethylene glycol molecules are attached to the interferon. The administration of pegylated interferon in combination with ribavirin increases the half-life of interferon and has the advantage of decreasing the frequency of dosing, hence patient compliance. Such treatment however has proven to be efficacious in less than 50% of treated patients. Given the increasing number of chronic sufferers of HCV, there is a need to develop a vaccine for both prophylactic and therapeutic purposes.
Development of a successful vaccine to protect against HCV infection has been elusive. One proposed reason for this difficulty is that HCV, being an RNA virus, is genetically unstable allowing it to achieve a high rate of viral mutation to evade the body's immune response. It is therefore a challenge for researchers to identify a portion of the virus that is conserved.
HCV has been classified in a separate genus (Hepacivirus) of the Flaviviridae family. HCV is non-cytopathic and rather triggers an immune response that either rapidly clears the infection or initiates an inflammatory response leading to chronic infection and liver injury. Spontaneously resolving infections that permanently clear HCV RNA without treatment occur in ˜30% of acute cases suggesting a natural immunity to HCV and is thus encouraging for the prospect of vaccine development. However, the determinants for this outcome of HCV infection are unknown.
The HCV virion contains a positive-sense single stranded RNA genome of about 9.5 kb. The genome encodes a single polyprotein of 3,010 to 3,030 amino acids. The structural proteins comprise a core protein forming the viral nucleocapsid and two envelope glycoproteins, E1 and E2. Some recent efforts towards the development of a HCV vaccine have focused on HCV envelope glycoproteins E1 and E2. It has been found that E1 and E2 form non-covalently associated heterodimers on the surface of the virion that mediate both viral attachment and entry and thus present targets for the host immune response.
Recent studies have suggested that envelope glycoprotein E2 binds to CD81 on the surface of CD4+ T cells. During the binding process, E2 undergoes rapid conformational change. To date no research has been able to provide a suitable modified envelope glycoprotein, which can exhibit “wild-type” levels of CD81 binding.
Throughout this specification, including the claims, all numbering of polypeptide residues of the HCV envelope glycoproteins E1 and E2 is based on the prototype HCV-H77 polyprotein sequence, Genbank Accession No. AF 009606. The mature form of glycoprotein E1 is encompassed by polyprotein residues 191 and 383, and the mature form of glycoprotein E2 is encompassed by polyprotein residues 384 and 746.
The receptor-binding domain (RBD) of E2 is encompassed by polyprotein residues 384-661 (E2661). Recombinant forms of E2661 RBD are efficiently secreted from transfected cells and are able to interact with CD81 and other cell surface molecules. The E2 RBD contains two variable regions, HVR1 (384-410) and HVR2 (474-482).
Variable region 1, located at the N-terminus of E2, is the most variable region in the HCV genome, is highly immunogenic and rapidly accumulates neutralization escape mutations. Despite the high level of amino acid variability in HVR1, there is an overall conservation of basic residues that are important for viral entry.
Variable region 2 is located within the region flanked by Cys-459 to Cys-486. Although originally described as a 7-residue sequence, comparison of E2 sequences from different HCV genotypes suggests it may extend from residues 461-481. In comparison to HVR1, the sequence of HVR2 is relatively stable within HCV infected people, although an accumulation of mutations at this location has been shown to correlate with responsiveness to interferon-α treatment.
In work leading to the present invention, the inventors have observed that alignment of E2 sequences representing the six major genotypes of HCV reveals a previously undescribed variable region between polyprotein residues 570-580 that is relatively conserved within a genotype but varies across genotypes due to amino acid insertions and deletions. Accordingly, amino acids 570-580 have been denoted the intergenotypic variable region (igVR). Examination of the corresponding region from all 6 genotypes of HCV, and divergent isolates therein, show that igVR is also flanked by conserved cysteine residues (Cys-569 and Cys-581), suggesting that these sequences form disulfide-constrained loops.
To date no vaccine treatment for HCV using the adaptive immune response route has been successful. Given the drawbacks of current and experimental therapies for treatment of HCV, there is an unmet need for providing a cell-mediated immune response to treat HCV infection.
It is one object of the present invention to provide an immunotherapeutic approach to prevent or treat HCV infection. A further object of the present invention is to provide an immunotherapeutic approach to prevent or treat HCV infectiona. A further object of the present invention is to provide a modified E2 glycoprotein, which approaches ‘wild-type’ binding levels to natural cellular receptors of HCV infection.
International Patent Publication No. WO 02/22155 (Hawaii Biotechnology Group, Inc.) discloses a truncated HCV E2 polypeptide which lacks the HVR1 region and is capable of secretion into growth medium when expressed in recombinant form in a host cell. The polypeptide may also lack its C-terminus after residue 662. International Patent Publication No. WO 03/022880 (XTL Biopharmaceuticals Ltd.) also discloses a truncated version of the E2 protein lacking HVR1 region.
The foregoing discussion is intended to introduce the field of the present invention and should not be construed in any way an admission of the state of common general knowledge in this art. Bibliographic details of publications referred to in this specification are set out at the end of the description. The reference to any prior art document in the specification is not, and should not be taken as, an acknowledgement or any form of suggestion that the document forms part of the common general knowledge.